The long term goals of this project are to understand how the activity, regulation and interactions of DNA topoisomerases control DNA topology and affect vital cellular functions. Drugs that initiate cell killing by trapping the covalent cleavage complex formed by type IB and type IIA topoisomerases are useful anti-cancer and anti-bacterial drugs. Previous results have shown that accumulation of type IA topoisomerase I cleavage complex can trigger bacterial cell death but specific inhibitors of type IA bacterial topoisomerase I remain to be discovered. The proposed research activities for the next funding period are relevant for both overcoming the critical barrier in the understanding of the mechanism of type IA topoisomerases and discovery of novel drugs targeting this class of topoisomerases. Topoisomerase I catalyzes the relaxation of DNA by cleaving a single strand in duplex DNA and passing the complementary strand through the break before religation of the cleaved strand. The molecular basis for the presence of a C nucleotide at the -4 position relative to the identified sites of cleavage by topoisomerase I has been revealed in the recently obtained crystal structure of the covalent cleavage complex. Site-directed mutagenesis and biochemical analysis will be used to test the hypothesis that interactions with this C nucleotide are important for relaxation of supercoiling catalyzed by E. coli DNA topoisomerase I. The results will reveal if the conservation of the C-nucleotide recognition during evolution is linked to the efficiency of the DNA winding activity required for physiological function. The molecular mechanism of how the passing DNA strand is guided by the enzyme during strand passage remains the critical barrier for progress in the understanding the overall enzyme catalytic mechanism. Partial DNA duplex molecules that are cleaved by E. coli topoisomerase I at a single site on one strand will be used for as substrates for determination of crystal structure of the full length enzyme as well as for mapping of site-specific protein-DNA interactions on the passing strand. The results will provide the molecular basis of how bacterial topoisomerase I catalyzes the important function of removal of excess negative supercoils by passing the complementary strand through the break after initial cleavage of a single strand. Genetic studies will test the hypothesis that certain perturbations of the non-covalent protein-DNA interactions observed in the crystal structure to be responsible for holding the 3'-OH portion of the cleaved DNA substrate in position for DNA religation could result in accumulation of DNA cleavage intermediate, leading to not only loss of topoisomerase I relaxation activity, but also potentially triggering the bacterial cell death pathway. Mutations in recombinant E. coli or Y. pestis topoisomerase I that can result in such perturbations of the non-covalent interactions with DNA will be identified by genetic selection for dominant lethal effects in E. coli and characterized biochemically. Success in these experiments would provide very useful information for discovery of novel antibacterial drugs targeting topoisomerase I specifically. PUBLIC HEALTH RELEVANCE: This project studies the structure and mechanism of bacterial topoisomerase I relevant for discovery of specific inhibitors. The proposed work would greatly aide the development of new antibacterial compounds targeting topoisomerase I as part of the much needed arsenal to combat the global health problem of multi-drug drug resistant bacterial pathogens.